Chemicals - Test method of two-generation reproduction toxicity study
Some standard content:
Ics13.300,11.100
National Standard of the People's Republic of China
GB/T 21758—2008
Chemicals--Test method of two-generation reproduction toxicity study2008-05-12Promulgated
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaAdministration of Standardization of the People's Republic of China
Implementation on September 1, 2008
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GB/T 21758—2008
This standard is equivalent to the Organization for Economic Cooperation and Development (OECD) Chemical Testing Guidelines No. 416 (2001) "Two-generation reproduction toxicity study" (English version).
This standard has been edited as follows:
---The scope section has been added;
The measurement units have been changed to the legal measurement units of my country; -The reference section of OECD has been deleted.
This standard was proposed and approved by the National Technical Committee for Standardization of Dangerous Chemicals Management (SAC/TC251). The responsible drafting unit of this standard is the Institute of Occupational Health and Poisoning Control, Chinese Center for Disease Control and Prevention. The co-authors of this standard are: Ningbo Entry-Exit Inspection and Quarantine Bureau, Guangdong Entry-Exit Inspection and Quarantine Bureau. The main drafters of this standard are: Sun Jinxiu, Chen Xiaoqing, Ma Zhongguan, Long Zaihao, Lin Zhenxing, Xu Lehui, Qiang Shujun. GH/T 21758—2008
OECD Introduction
1. In June 1995, the OECD Reproductive Development Working Group held a discussion meeting in Copenhagen, requiring the current OECD reproductive developmental toxicity test guidelines to be updated and new developmental toxicity test endpoints not included in the original guidelines to be developed. The working group recommended that the two-generation reproductive toxicity test should be revised based on the proposals of the United States and Germany. The working group reached a consensus on all major revisions to this guideline. 2. Provide comprehensive data on the effects of the test samples on the entire reproductive function and behavior of the reproductive system of female and male animals: such as gonadal function, estrous cycle, mating behavior, conception, pregnancy, delivery, lactation, weaning, and the growth and development of offspring, etc., and also provide preliminary data on neonatal diseases, deaths, and prenatal and postnatal developmental toxicity, etc., to provide reference for the next step of toxicity testing. In addition to studying the growth and development of F1 offspring, this test can also systematically evaluate the reproductive and behavioral growth and development of the entire reproductive system of F2 offspring and F2 generation females and males. In order to obtain more comprehensive information on developmental and functional defects, it may sometimes be necessary to conduct additional developmental and/or developmental neurotoxicity tests or study these endpoints in other tests using other test guidelines. 1 Scope
Test methods for two-generation reproductive toxicity of chemicals
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GB/T21758—2008
This standard specifies the scope, basic test principles, test methods, test data and reports of two-generation reproductive toxicity tests of chemicals. This standard is applicable to the detection of reproductive toxicity of chemicals. 2 Basic test principles
2.1 In this test, male and female animals are divided into different dose groups with a certain gradient. Male mice of the P generation should be exposed to the test substance during the growth period and at least cover a complete spermatogenesis cycle (about 56 days for mice and 70 days for rats) to induce adverse effects on the spermatogenesis process. The effects of the test substance on sperm can be detected by measuring sperm counts (e.g., sperm morphology and sperm motility) and tissue preparations and detailed histopathological examinations. If data on sperm production can be obtained from repeated burial experiments that have been completed for a sufficiently long test period (e.g., 90 days), it is no longer necessary to evaluate the male parent (P) in this test. However, for future evaluations, it is recommended to save sperm samples and record data from the male parent (P). In order to evaluate the effects of the test substance on estrus, P generation dormice should be induced from the growth period and continue to mate for several complete estrus periods. Both males and females continue to be induced during the mating period. Parent mice (P) should continue to be induced during pregnancy and until the offspring F1 is weaned. The test substance should continue to be induced after the offspring F1 is weaned, and from the growth period, maturity, mating, birth of the offspring F2 of the offspring F1, until the offspring F2 is weaned. 2.2 All animals should be observed clinically for toxic symptoms and pathological examinations, with special attention paid to the overall functional status and performance of the reproductive system of male and female animals and the effects on the growth and development of offspring. 3 Test methods
3.1 Test preparation
3.1.1 Selection of animal strains
Rats are preferred. If other strains are used, appropriate reasons should be given and some necessary changes should be made. However, strains with low fertility or known developmental defects should not be used. Before the start of the test, the weight variation between test animals should be reduced as much as possible so that it does not exceed 20% of the average body weight of the alternate sex.
3.1.2 Housing conditions
The temperature of the test animal room should be maintained at 22℃±3℃. Although the relative viscosity should be at least between 30% and 70%, it is best to control it at 50% to 60%. Artificial lighting should be used, with 12h alternating light and dark. The animals should be fed with routine simple feed in the laboratory, and drinking water is not limited. When the feed is contaminated, it should be ensured that the test substance is mixed evenly in the feed.
The experimental animals can be kept in single cages or in groups of small numbers according to sex. During the mating process, the female mice should be taken from the feeding cage and placed in a suitable cage to facilitate confirmation of the mating situation. After mating is confirmed, the female mice should be placed in the farrowing cage or the farrowing cage for single breeding; they can also be kept in groups of small numbers and moved to the farrowing cage for single breeding 1 or 2 days before farrowing. When farrowing is approaching, the pregnant mice should be provided with appropriate bedding. 3.1.3 Animal preparation
Select young, healthy animals that have not undergone other experiments. Acclimate to the laboratory environment for at least 5{ before the experiment. The experimental animals should be marked with the breed, strain, source, sex, weight and (or) age. The relatives of the animals used should be clarified before the experiment to avoid sibling mating. The animals should be randomly divided into pairs and treatment groups (randomization by weight is recommended). The cage placement should also be as far as possible to avoid the impact of different resting positions on the experiment. Each animal should be individually marked. Parent (P) animals should be treated before the start of exposure. For offspring FI, it should be done before the animals are weaned and selected for mating. All selected offspring FI animals should record and save their litter information. When mice need to be weighed or undergo certain tests, they should be marked with a unique mark as early as possible after birth. Parent (P) animals should be exposed from 5 to 9 weeks after weaning. All experimental group animals should be as close in size and age as possible. GB/T 21758--2008
3.2 Experimental procedures
3.2.1 Gender and number of animals
There should be enough animals in the exposed and control groups to ensure that 20 pregnant mice are included before delivery. For substances that cause infertility after exposure, excessive toxicity in high-dose groups, death, etc., it is sometimes not possible to obtain 20 pregnant mice. Only with a sufficient number of pregnant mice can we make a valuable assessment of fertility, conception, maternal behavior, lactation, and the potential effects on the growth and development of the F1 offspring from the fetus to maturity and the birth to weaning of the F2 offspring. Therefore, if the expected number of pregnant animals (e.g., 20) cannot be obtained, the test results are not necessarily invalid, but should be analyzed on a case-by-case basis.
3.2.2 Preparation of test substances
It is recommended to use the oral route for test animals (e.g., mixing into feed, drinking water, or oral gavage). If necessary, other routes of administration (e.g., percutaneous or inhaled) can also be used.
The test substance should be dissolved or suspended in a specified solvent. It is recommended that aqueous solutions or aqueous suspensions be used first, and oil solutions or emulsions (e.g., corn oil) and other solvents should be considered. When using other solvents, the toxicity of the solvent should be understood and the stability of the test substance in the solvent should be determined.
3.2.3 Dose design
At least three dose levels should be set and
parent animal mortality
toxicity should not be caused
and should be controlled below 10%
the two components
adverse effect level (NOAEL or,
distance is usually selected from 2 to 4
feed test,Dose group! Feed. Whether the test substance or its counterpart is suitable. If the strategy should be set, the weight should not exceed the metabolic and animal weight of the subject. The control group should not be treated with any load. If the animals in the control group are treated in the same way as the brain, and the solvent is given to reduce the rate of bacterial use, it is necessary to consider the toxicity characteristics of the solvent and other substances to the test substance; and for the level of the limit test in 3.2.4, the dose of silver should be read. All information on the maintenance of the group should also be considered. If the male is stable. However, due to the limitation of herd effect, the mortality of the selected highest dose was not related to the exposure and the baseline dose (BMD) was not observed. The decrease between groups was very large (for example, more than 10 times). In the oral toxicity study of the test substance, at least one dose is higher than 1000 mg/(kg·d). If no toxic effect is observed when the test substance is exposed to the test substance through the operation specified in this test or through feed and drinking water, or if the data on related compounds based on structure and/or metabolism show no toxic effect, then a complete test of the test substance is not necessary. If the human exposure data show that a higher oral dose is required, then the limit test should not be used. For other routes of administration, such as inhalation or dermal administration, reference is often made to the physicochemical properties of the test substance, such as solubility, which can often provide the highest concentration for the drug to be administered. 3.2.5 Administration Procedures
Oral administration (feed, drinking water or oral gavage) is preferred. 7. If other routes of administration are used, justification should be provided and appropriate adjustments should be made. All animals must be sampled in the same manner throughout the study. If the test substance is administered by oral gavage, a gastric tube should be used. The volume of each gavage should not exceed 1 mL/100 g (if rice oil is used, the maximum volume is 0.4 mL/100 g); if it is an aqueous solution, the maximum volume can be increased to 2 mL/100 g. During the test, except for the concentration of irritants or corrosive substances that will aggravate the effect at high concentrations, the concentration should be adjusted to minimize the variability of the test volume to ensure that the volume is constant at all dose levels. If the test substance is administered by gavage, ensure that the lactating rats ingest the test substance indirectly through milk and that the test substance is not administered directly until they are weaned. When the test substance is administered by gavage, the rats can ingest the test substance in the late lactation period when the rats begin to feed themselves.
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GB/T 21758-2008
When the test substance is administered by gavage, the most important point is to ensure that the proportion of the test substance added does not disrupt the normal nutritional and water balance of the animals. When the test substance is administered by gavage, the exposure level can be expressed as a constant concentration (g/g) or as a constant dose level calculated based on the body weight of the animals; whichever is chosen, detailed instructions should be provided. When the test substance is administered by gavage, the dose should be administered at the same time each week, and the dose should be adjusted at least once a week based on the body weight to maintain a constant level. When adjusting the gavage dose based on body weight, the distribution of the test substance in the pan should be considered. 3.2.6 Test Procedure Www.bzxZ.net
Parent (P) female and male rats should be exposed to the test substance from 5 to 9 weeks of age. F1 female and male rats should be exposed to the test substance from the time of weaning. It should be noted that if the test substance is given through feed or drinking water, the F1 generation may have been exposed to the test substance during lactation. Both parent (P) and F1 male rats should be exposed to the test substance for at least 10 weeks before mating. The 2-week mating period should also be exposed to the test substance. Male rats that are no longer needed to evaluate reproductive effects should be humanely killed and dissected. Parent (P) female rats should be exposed to the test substance from the gestation period until the F1 generation is weaned. If there is data on the affinity, metabolic induction or bioaccumulation of the test substance during this period, the exposure plan should be adjusted accordingly. In general, the individual doses should be determined based on the appropriate weight loss of each animal, but dose adjustments during pregnancy should be particularly cautious.
Parents and offspring should be
parents and F1 female and male mice, and all offspring should be kept.
, and the animals should be killed humanely.
3.2.7 Animal mating
3.2.7.1 Parent mating
Each time, the mice should be kept with a sterile ... 3.2.7.2 Offspring mating F1 offspring mating should be performed at weaning time to prepare F2 offspring. If there is a problem, select animals from the litter for questioning. It is best to select according to the fallow period, but not to exclude the unpregnant pair. The unpregnant animals should be kept in the same cage (i.e., paired) until conception or at the longest day of pregnancy. If mating fails, re-mating should be performed. The paired animals should have clear only and no significant difference with other animals of the same dose of leukemia. Representative animals should be selected randomly from each litter. From a practical point of view, the most mature animals can be re-mated.
The animals that have been proven to be viable should be mated again for a second time to check for pathological changes in reproductive organs and the function of the estrus cycle. 3.2.7.3 Second mating
Sometimes, if the litter size and condition-related changes found during the first mating are not clearly determined, it is recommended that the parental P or F1 animals be mated again to produce a new offspring. This is done by mating a female or male that has not produced offspring with a rat of the opposite sex that has been proven to be fertile. If the parental P and F1 generations are to be bred for the second time, the second mating should be carried out about one month after the last litter is weaned.
3.2.8 The litter size
should allow the animals to reproduce normally and to nurse offspring to weaning. Standardization of the case is difficult, but the method is not clearly defined; when it is standardized, the method used should be described in detail.
3.3 Observation Results
3.3.1 Clinical Observation
Clinical observation must be conducted daily. In the oral gavage test, the observation time should be during the expected peak of the reaction after infection. The behavioral changes, signs of dystocia or delayed labor, and all signs of the animals should be recorded in detail. In addition, a detailed examination should be conducted at least once a week, preferably when the examination is performed during weighing. In addition, the animals should be observed twice a day (once on weekends) for morbidity and mortality. 3.3.2 Body weight and food/food consumption of the parents In the oral gavage observation, the parents (P and F) should be weighed on the day of the initial exposure and at least once a week thereafter. Parent females (P and F1)3
GB/T 21758-2008
should be weighed on days 0, 7, 14 and 20 or 21 of gestation, and on the day of litter weight measurement and the day of sacrifice during lactation. These observations should be reported for each adult animal. Minimum food intake should be measured at least weekly during the pre-mating period and during gestation. If the test substance is given in drinking water, water consumption should also be measured at least once a week. 3.3.3 Estrus cycle
The duration and status of estrus in P and F1 females can be evaluated by vaginal smear observations before and at any time during mating until evidence of successful mating is obtained. If vaginal/cervical blasts are found in the vaginal smear, it is necessary to distinguish whether it is caused by mucosal dysplasia or pseudopregnancy.
3.3.4 Sperm parameters
At the end of the experiment,The testicular and testicular mass of all P and F1 males should be recorded and one uterus fixed for histopathological examination. For at least 10 males per group of P and F1 males, unfixed testes and epididymis should be used to determine germ cell homogenization and cauda epididymal sperm counts. Sperm collected from the cauda epididymis or vas deferens should be evaluated for sperm motility and sperm morphology. If treatment-related effects are observed or if the test substance has been shown to have an effect on spermatogenesis in other studies, sperm evaluation should be performed on all males in each dose group; otherwise, sperm counts should be limited to control and high-dose P and F1 animals. The total number of testicular homogenized spermatocytes and cauda epididymal sperm should be counted. The cauda epididymal sperm stock can be calculated by counting the sperm concentration and volume in the suspension for qualitative evaluation or by recovering sperm from the remaining cauda epididymal tissue in the crotch or pulp. Males selected from all dose groups should be counted immediately after sacrifice, unless video or digital records have been made or specimens have been frozen for later analysis, in which case the highest dose group and the control group should be analyzed first. If no treatment-related effects are seen in the highest dose group (e.g., studies of sperm count, sperm motility, and sperm morphology), then no further analysis of the other dose groups is necessary. Conversely, if treatment-related effects are found in the most distant dose group, then the other dose groups should be further evaluated. Epididymal (or vas deferens) sperm motility should be assessed or videotaped immediately after sacrifice. Sperm should be recovered with minimal trauma and diluted for motility analysis using an acceptable method. The percentage of motile spermatozoa may be determined subjectively or objectively. If computer-assisted motility analysis is used, sperm motility is determined based on user-defined mean passage velocity and linear or linear index readings. If samples were videotaped or photographed at the time of dosing, only the control and high-dose F1 animals should be analyzed subsequently; if treatment-related effects are observed, the other low-dose groups should also be evaluated. If no videotaping or photographing was done, samples from all dose groups should be analyzed at dissection. Sperm morphology should be analyzed in the vas deferens (or vas deferens). Sperm should be examined in fixed, swollen samples (at least 200 per sample) and classified as normal or abnormal. Abnormal sperm morphology includes sperm fusion, free heads, morphological heads, and () malformed sperm. Sperm morphology should be analyzed immediately after sacrifice of selected males from each dose, or videotaped or photographed for later analysis. Smears can also be reviewed later after fixation. In these cases, the highest dose group and control group should be analyzed first, and if no treatment-related effects (e.g., effects on sperm morphology) are found in the highest dose group, no other dose groups need to be analyzed. Conversely, if the highest dose group shows a treatment-related effect, the other dose groups should also be analyzed and evaluated. If all the above parameters have been examined in the sperm in a systematic compliance study of 90 days or more, it is not necessary to repeat the measurements in the two-generation reproduction study. However, it is recommended to save samples of parental sperm or digitally record them for later evaluation if necessary. 3.3.5 Offspring
should be examined as soon as possible after parturition (the day of lactation), including the number of pups, sex, stillbirths, live births and abnormalities. If dead pups are found on the day of parturition, check for defects, find out the cause of death and make recommendations. The number of live pups should be counted and weighed individually on the day of birth (the day of lactation) or the first day after birth, and weighed on the 4th, 7th, 14th and 21st days of lactation. At the same time, any observed abnormal physical behavior of the mother and the offspring should be recorded. The physical development of the offspring is mainly recorded by weight gain. Other physical parameters (e.g. ear opening, chin opening, teething, hair growth) can provide additional information, but this information is best evaluated together with sexual maturity data (e.g. age and weight at vaginal opening or prepuce separation). If no separate tests are designed for related functions (e.g. motor activity, sensory function, development of reflex function), especially functional tests related to sexual maturity, these functions should be tested before and/or after weaning in the F1 offspring: the vaginal opening and prepuce separation of the offspring selected for mating should be checked after weaning. If the offspring's color changes from that at sexual maturity, the distance between the anus and genitals should be measured on the day of the second offspring. If animals show obvious adverse effects (e.g. significantly slow weight gain, etc.), these groups can be exempted from functional tests. If functional tests are performed, the offspring that will be mated should not be selected. 3.3.6 Gross Anatomy
All parental (P and F1) animals that died at the end of the experiment and during the experiment, all offspring with deformities or clinical symptoms, and the litters discarded from the 1st and 2nd generations (one female and one male per litter of each sex) should be observed with the naked eye for structural abnormalities and pathological changes, especially morphological abnormalities or pathological changes in the reproductive organs. The mice that died or were humanely killed due to necrosis should also be examined for defects and (or) causes of death before being immersed and fixed, and the labels should be preserved. Inspect the uterus of all primiparous females and observe the number of implanted mice. 3.3.7 Organ Weights
At the end of the experiment, all P and FI animals should be weighed and the following organ weights measured (paired organs should be weighed separately): uterus: ovary; testicles, epididymis (entire epididymis and cauda epididymis); prostate; seminal vesicles (containing the coagulation glands) and their carboxylates (as a unit); brain, liver, kidneys, spleen, pituitary, thyroid, adrenal glands and known target organs. At the end of the experiment, the rats selected for gross anatomy in F and FI should be weighed and the brain, spleen and thymus of one randomly selected animal of each sex per litter should be weighed. If feasible, the significance of gross anatomy and organ weights should be evaluated in conjunction with the observations of other replicates. 3.3.8 Histopathological Examination
3.3.8.1 Parent Animals
The following organs and cysts of parent and offspring should be fixed and preserved for microscopic examination.
-Vagina, uterus with cervix and ovum (preserved in appropriate fixative) one side of the ovum (preserved in Bouin's or similar fixative) one side of the ovum, spermatogonia, prostate and coagulation glands; all identified target organs in all P and F animals selected for mating. Surface histopathological examination of the organs and tissues listed above should be performed on all F1 and F1 animals selected for mating in the high-dose and control groups, and ovaries of parental P animals as an alternative. Sometimes, in order to better clarify the NOAEL, the organ should also be examined in the low-dose and medium-dose groups when changes in the organ are shown to be related to the treatment. In addition, histopathological examination of the uterus of low-dose and medium-dose animals suspected of reproductive impairment (e.g., inability to mate, receptivity, inability to produce or secrete normal offspring, or changes in estrous cycle, sperm quantity, motility or morphology) should be performed. All macroscopic lesions, atrophy or tumors should be examined.
The testicles should be retrogradely examined with detailed histopathology (e.g., fixation with Bauins solution, stone embedding, and 4-5 μm thick sections) to identify treatment-related effects, retained sperm cells, missing germ cell layers or unidentified types, and multinucleated spermatocytes or spermatogenic succession cells in the seminal cavity. The entire epididymis should be examined, including the head, body, and tail, and can be examined through the section of the epididymis. The epididymis should be evaluated based on leukocyte infiltration, degree of cell type changes, abnormal cell types, and the state of sperm engulfment. Male genitalia can be stained and examined with para-aminosalicylic acid (PAS) and hematoxylin. Histopathological examination of the postlactating ovary should include the initial and growing follicles and the corpus luteum during lactation. The histopathological changes should be qualitatively determined by the number of primitive follicles. The F1 females should be subjected to a quantitative analysis of primordial follicles; the number of animals, the choice of ovarian sections, and the size of the sections should be consistent with the statistical requirements of the evaluation procedure used. This analysis should include counting of primordial follicles (which are often mixed with small growing follicles) and comparing the numbers of infected and control groups.2. All pups with external defects or clinical symptoms, or one pup of each sex randomly selected from the F1 and F2 generations that have not been selected for mating, should be examined for abnormal tissues and organs, fixed and preserved for histopathological examination. When performing histopathological examination of the tissues, special attention should be paid to the organs of the reproductive system. 5
Weight and absolute and relative organ weights of the parent animals sacrificed; e
) Clinical symptoms, severity and duration (whether they are reversible); g) Time of death during the test or whether the animal survived to the end of the test; GB/T 21758--2008
V) Toxic reactions recorded separately by sex and dose group, including mating success rate, fertility, conception rate, live birth rate, birth rate, fertility rate and milk rate; the report should include the number of animals used to calculate these indices; degenerative or other effects on reproduction, offspring and postnatal growth; gross anatomical findings!
Detailed summary of all histopathological findings; number of P and F1 females with normal cycles and cycle length; total number of spermatozoa attached to the labia, percentage of sexually active spermatozoa, percentage of morphologically normal spermatozoa and percentage of each abnormality;
mating, duration of mating, including days before mating; length of gestation,
number of follicles, corpora lutea and fetuses;
g) number of litters and postimplantation losses;
if dysplastic pups are counted, the number of pups with gross abnormalities should be reported; physical data on pups and other postnatal development, with an explanation of the physical parameters evaluated;
t) functional examination data on pups and adults, if available; appropriate statistical treatment of the results.
4. 3. 6 Discussion of results.
4.3.7 Conclusion, including NOAEL values for the effects on mother mice and pups. 4.4 Interpretation of test results
This test can reflect the effects on the reproductive function of animals after multiple exposure to a test sample throughout the reproductive cycle. In particular, the test can provide information on reproductive function parameters, offspring growth and survival. When interpreting the test results, a comprehensive analysis should be conducted in combination with the results of subchronic tests, teratogenic tests, case dynamics tests and other tests. It can often be used to evaluate whether the chemical needs further research, but there are still certain limitations in extrapolating the test results to humans. The test results can provide the no-effect dose level and the basis for establishing the safe exposure level for human beings:
GB/T21758-2008
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National Standard of the People's Republic of China
Test method for two-generation toxicity of chemicals
GB/T 21758—2008
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